| Part I Establishment of the mimetic aging rat model induced by D-galactoseObjective:To establish the mimetic aging rat model induced by D-galactose (D-Gal).Methods:Forty-eight2-month-old female Sprague Dawley (SD) rats were randomly divided into two groups:a control group (NS group) and a D-Gal group (mimetic aging group). The rats in the D-Gal group were injected subcutaneously with D-Gal (500mg/kg/d) for8weeks; the rats in the control group were injected with the same volume of vehicle (0.9%saline) on the same schedule. After the last injection, both the NS and D-Gal groups were divided into3age subgroups:4-month-old (just after the injection),10-month-old (6months after the last injection), and18-month-old (14months after the last injection). Then the auditory function was examined by auditorybrainstem response (ABR); the ultrastructural morphology in the auditory cortex was examined using a Transmission Electron Microscope; the density of neurons was tested by toluidine blue staining and the cell apoptosis in the auditory cortex was tested using terminal deoxynucleotidyltransferase-mediated UTP nick-end labeling (TUNEL). Results:Compared with the age-matched NS group,10-and18-month-old D-Gal groups showed attenuated high-frequency hearing loss. Compared with the age-matched NS group, the abnormal ultrastructure of auditory cortex neurons was more significant in the D-Gal group. Irregular nuclear, condensed chromatin, accumulated lipofuscin, swollen mitochondria and disrupted myelin were found in D-Gal group much earlier than age-matched NS group. Compared with the age-matched NS group, the density of auditory cortex neurons in18-month-old D-Gal group was significantly reduced (P<0.05). Besides, the number of TUNEL-positive cells was significantly increased in the10-and18-month-old D-Gal groups compared with the age-matched NS groups (P<0.01).Conclution:The degeneration of neurons in auditory cortex, the reduced density of neurons and cell apoptosis accompany normal aging. Chronic exposure to D-Gal accelerates the development of cell senescence in the auditory cortex and promotes the occurrence of presbycusis. Part II Relationships between telomeres, telomerase and aging auditory systemObjective:To investigate the possible relationships between telomeres, telomerase and aging auditory system.Methods:Seventy-two2-month-old female Sprague Dawley (SD) rats were randomly divided into two groups:a control group (NS group) and a D-Gal group (mimetic aging group). The rats in the D-Gal group were injected subcutaneously with D-Gal (500mg/kg/d) for8weeks; the rats in the control group were injected with the same volume of vehicle (0.9%saline) on the same schedule. After the last injection, both the NS and D-Gal groups were divided into3age subgroups:4-month-old (just after the injection),10-month-old (6months after the last injection), and18-month-old (14months after the last injection). Then ROS in the plasma was tested by Colorimetry; telomere length and TERT mRNA expression were performed using RT-PCR; TERT protein level was tested by western blotting.Results:Compared with the age-matched NS group, the levels of H2O2and MDA in the plasma were significantly increased in the10-and18-month-old D-Gal groups (P<0.05), the activity of T-SOD was significantly decreased in the10-and18-month-old D-Gal groups (P<0.05); telomere length was significantly decreased in the18-month-old D-Gal group (P<0.05); the expression of TERT mRNA was significantly decreased in the4-,10-and18-month-old D-Gal groups (P<0.05); the level of TERT protein was significantly reduced in the10-and18-month-old D-Gal groups (P<0.01).Conclusion:Telomere length and telomerase activity were negatively regulated by age; the decrease of telomere length and TERT expression were accelerated in D-galactose-induced aging auditory system. Part â…¢ The role of mitochondrial sirtuin deacetylase Sirt3in the D-galactose-induced aging auditory cortexObjective:To investigate the mechanism of Sirt3in the development of D-galactose-induced aging auditory cortex.Methods:One hundred and forty-four2-month-old female Sprague Dawley (SD) rats were randomly divided into two groups:a control group (NS group) and a D-Gal group (mimetic aging group). The rats in the D-Gal group were injected subcutaneously with D-Gal (500mg/kg/d) for8weeks; the rats in the control group were injected with the same volume of vehicle (0.9%saline) on the same schedule. After the last injection, both the NS and D-Gal groups were divided into3age subgroups:4-month-old (just after the injection),10-month-old (6months after the last injection), and18-month-old (14months after the last injection). Then MDA concentrations and SOD2activity were tested by Colorimetry; the deletion of mtDNA4834bp was evaluated using Taqman RT-PCR; Sirt3and SOD2mRNA expression were tested by RT-PCR; Sirt3and SOD2protein levels were examined by western blotting; the acetylation level of SOD2was evaluated by immunoprecipitation and western blotting; the expression of Sirt3protein was performed by immunofluorescence assay.Results:Compared with the age-matched NS group, MDA concentration in the4-,10-, and18-month-old D-Gal group was increased by1.18-,1.39-(P<0.05) and1.32-fold (P<0.05), respectively; SOD2activity was reduced by1.21-,1.36-(P<0.05) and4.50-fold (P<0.01), respectively; accumulation of the CD was increased by1.51-(P<0.01),1.87-(P<0.01) and1.90-fold (P<0.01), respectively; Sirt3mRNA expression was reduced by1.32-(P<0.05),1.70-(P<0.01) and4.46-fold (P<0.01), and the SOD2expression was reduced by1.06-,1.11-(P>0.05) and2.04-fold (P<0.01), respectively; the protein levels of Sirt3were reduced by1.46-,2.10-and3.47-fold (P<0.01), and those of SOD2were reduced by1.03-,1.10-(P>0.05) and2.27-fold (P<0.05), respectively; the acetylation level of SOD2was increased by2.70-,3.09-and3.31-fold (P<0.01), respectively.Conclusion:Decreased Sirt3expression in the D-galactose-induced aging auditory cortex may have less ability to deacetylate SOD2, resulting in a reduction in SOD2activity and an elevation in ROS generation. As a result, oxidative stress accumulated with aging, mitochondria dysfunction, abnormal ultrastructural changes and auditory cortex cell apoptosis occur, which ultimately results in AHL. |
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